Prevalence of sensitization to molecular food allergens in Europe: A systematic review

Abstract Background Recent reports indicate that the prevalence of food allergy is increasing, but accurate estimates remain a challenge due to cross‐reactivity and limited use of precise diagnostic methods. Molecular allergy diagnostics, in which sensitization to individual molecular allergens is measured, is emerging as a promising tool for evaluation of sensitization profiles. In this systematic review, we summarized estimates of prevalence of sensitization to molecular food allergens in the general population in Europe. Methods Following a protocol prospectively registered with the International Prospective Register of Systematic Reviews (PROSPERO; reference CRD42021266657), we searched seven databases with no restrictions on publication date or language. Two reviewers independently screened the literature, extracted data, and appraised the risk of bias in the included studies. The findings were synthesized narratively. Results From 4776 de‐duplicated records, five studies, with low to moderate overall risk of bias, were included. Forty‐six molecular allergens from 18 foods were investigated. Overall, the prevalence of sensitization was low, particularly for major allergens, and non‐existent for 10 molecular allergens (0% [95% CI 0–0.8]). The highest prevalence was seen for PR‐10 proteins, such as Cor a 1.04 (13.6% [95% CI 10.9–16.9]). Conclusions Available data, primarily from North‐western Europe, indicate that sensitization to molecular food allergens is overall low. The highest estimates were found for cross‐reactive PR‐10 proteins. There were not enough studies to discern regional differences or perform meta‐analysis, highlighting the need for more population‐representative studies in order to elucidate patterns of sensitization to molecular food allergens in Europe.

Results: From 4776 de-duplicated records, five studies, with low to moderate overall risk of bias, were included. Forty-six molecular allergens from 18 foods were investigated. Overall, the prevalence of sensitization was low, particularly for major allergens, and non-existent for 10 molecular allergens (0% [95% CI 0-0.8]). The highest prevalence was seen for PR-10 proteins, such as Cor a 1.04 (13.6% [95% CI 10.9-16.9]).
Conclusions: Available data, primarily from North-western Europe, indicate that sensitization to molecular food allergens is overall low. The highest estimates were found for cross-reactive PR-10 proteins. There were not enough studies to discern regional differences or perform meta-analysis, highlighting the need for more population-representative studies in order to elucidate patterns of sensitization to molecular food allergens in Europe. Assessed with the "gold standard" 5 method of double-blind placebocontrolled food challenge (DBPCFC), the estimate was 0.9% (95% CI 0.8-1.1). As DBPCFC is resource-intensive and time-consuming, this method is seldomly used in epidemiologic studies or outside of the academic context. [4][5][6][7] At the molecular level, foods such as peanut are composed of multiple components, some of which are classified as molecular allergens. 8 Similarity in structure and properties of molecular allergens can give rise to cross-reactivity between different foods, as well as between foods and other types of allergen sources, for example, inhalant allergen sources such as pollen and mites. 9 Cross-reactivity is commonly expressed with mild symptoms of the oral mucosa following ingestion of certain foods, but may also present with severe systemic reactions, depending on the cross-reactive allergen, such as lipid transfer proteins (LTPs). 10 Traditionally, in vitro diagnostics of food allergy has involved measurement of specific immunoglobulin E (sIgE) sensitization to whole allergen extracts. In contrast, molecular allergy diagnostics has enabled measurement of sensitization to individual molecular allergens. 11,12 Molecular allergy diagnostics is a promising tool to establish the source of primary sensitization and the origins and patterns of cross-reactivity. 11,[13][14][15][16] The advent of biochip technology, where sensitization to an array of molecular allergens can be tested simultaneously using small quantities of serum, 17,18 has further increased the utility of molecular allergy diagnostics, particularly in complicated cases 19,20 and to produce rich sensitization profiles. 12,20 As management of food allergy includes avoidance of foods, and in some cases resource-intensive immunotherapy, [21][22][23] it is important to establish correct diagnosis. In the last 2 decades, a large body of research on molecular allergy diagnostics has been produced. 12,24 However, to the best of our knowledge, no systematic review has been undertaken to synthesize the existing literature and elucidate the sensitization patterns to molecular food allergens across Europe in the general population. Therefore, in updating the previous EAACIcommissioned systematic review, 4 our aim in this systematic review was to estimate the prevalence of sensitization to molecular food allergens in the general population in Europe as defined by sensitization to molecular food allergens.

| Protocol
This systematic review was performed according to a protocol, which was reported according to the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P). 25 The protocol was prospectively registered in the International Prospec-

| Outcomes
Sensitization to any molecular food allergen, as assessed by sIgE above a study-defined threshold, using any molecular allergy diagnostic method. Studies which did not present data for each individual or the percentage of sensitized individuals were excluded.

| Search methods
Allergome, CINAHL, the Cochrane Library, EMBASE, MEDLINE, Scopus, and Web of Science were searched. These databases were selected to keep in line with previous systematic reviews on the topic as well as what has been previously suggested to attain a comprehensive coverage of relevant journals. 4,28 The databases were searched for articles published from inception up until June 30, 2021 (except Allergome, which was searched on September 1, 2021). The search queries were structured as four blocks concatenated with the "AND" Boolean operator. The search blocks contained, respectively, keywords for: 1) food allergy, foods, and molecular allergens; 2) epidemiological measurements (e.g., "prevalence"); 3) molecular allergy diagnostic-related terms (e.g., "molecular diagnos*"); 4) Europe and its countries. The search strategy was Allergome was searched by: 1) searching for the first molecular allergen (in alphanumeric order) of that food (e.g., "Ara h 1" for peanut); 2) browsing to the page for each molecular allergen of that food (e.g., https://www.allergome.org/script/dettaglio.php?id_mole-cule=50 for Ara h 1); 3) fetching articles in the "Epidemiology from Literature" table. Due to the overwhelming number of allergens in Allergome, steps 1)-3) were repeated only for the eight most common foods. 30,31 The search queries are detailed in Supporting Information S2.
Screening of title/abstract, full-text screening, and general management of full-text PDFs was done in Rayyan (https://rayyan.ai).

| Screening and selection
The first stage of screening was based on title/abstract. Irrelevant articles, and those meeting any exclusion criterion, were excluded. In the second stage, the full-text of each article was screened. This process was performed by two reviewers (DL and AI) independently.
After each stage, the decisions were unblinded and compared for differences, which were resolved through discussion. No arbitration through a third reviewer was required.

| Data extraction
A Microsoft Excel (Microsoft Corporation, 2021) extraction form (available upon request), piloted a priori, was used to extract data.
Two reviewers (DL and AI) extracted data independently, and differences were resolved through discussion. No arbitration through a third reviewer was required.

| Data items
The following data items were extracted and summarized from each study: surname of first author; publication year; study design; number of individuals recruited/included; period of data collection; country of study; subject characteristics (age, gender, and epidemiological/medical background); molecular allergen(s) investigated; diagnostic method and cut-off value(s); results (e.g. sensitization prevalence).

| Assessment of risk of bias
Assessment of risk of bias in the included studies was done using forms based on the Critical Appraisal Skills Programme (CASP) tool.
CASP checklists for systematic reviews, cohort studies (reused for cross-sectional studies), and case-control studies, respectively, were adapted. Specific questions differed somewhat between the forms for respective study design, but assessed the same four sections: study LISIK ET AL.
-3 of 13 design, participant selection, exposure assessment, and outcome assessment. Each of these sections were rated as "high", "moderate", or "low" risk of bias, or "not applicable"/"not available", depending on available information. Each study was also given an overall rating, based on the section ratings and a collected assessment. This process was performed by two reviewers (DL and AI) independently. Differences were resolved through discussion. No arbitration through a third reviewer was required.

| Data synthesis
A descriptive table was produced to summarize essential characteristics of the included studies. The results were narratively synthesized.

| Statistical analyses
Prevalence was defined as p ¼ n sensitized =n total . The 95% CI was estimated using Wilson's score interval without continuity correction, 36,37 with p lower ¼ 2r þ z 2 − z ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffiffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffiffi , where r = n sensitized , q = 1 -p, and z = 1.96 (the 97.5th percentile value from the standard normal distribution for α = 0.05).

| Deviations from the protocol
After having conducted the initial searches, the authors identified Allergome as a relevant source, housing articles not identified with previous methods. A search of Allergome was thus performed post hoc.

| Study selection and characteristics
Our searches yielded 5425 records. After de-duplication, 4776 records were screened. Of these, five studies [38][39][40][41][42] were included in the systematic review. Figure 1 illustrates the PRISMA flow diagram of this process. Across the five studies, 46 molecular allergens from 18 foods were investigated, of which peanut and wheat were the most frequently examined. One study 41 presented period prevalence estimates and time trends data, while the remaining data estimated point prevalence. There were two cross-sectional studies 40,42 and two cohort studies. 39,41 The fifth study 38 Table 1.

| Assessment of risk of bias
Two studies 40,41 were rated as having an overall "low" risk of bias, while three studies 38,39,42 were rated as "moderate" ( Table 2).

| Peanut
Molecular peanut allergens were evaluated in two studies. 38,42 Differences in sensitization prevalence are illustrated in Figure 3.
Asarnoj et al. 38 performed a nested case-control study within a Swedish birth cohort at the time of the eight-year follow-up, assessing sensitization to Ara h 1-3 and Ara h 8. Four groups, each consisting of 50 children, representing four different sensitization patterns to whole peanut and birch pollen, were randomly sampled.
They found point prevalence estimates of ≥42% to each of the major peanut allergens Ara h 1-3 in those sensitized to peanut but not birch pollen, apart from Ara h 8, to which none in this group were sensitized. In contrast, in the group sensitized to both peanut and birch pollen, sensitization was most common to Ara h 8 at 38% (95% CI 25.9-51.9), 8% (95% CI 3.2-18.8) to Ara h 1 and 3, respectively, and 36% (95% CI 24.1-49.9) to Ara h 2. In those not sensitized to neither birch pollen nor peanut, none were sensitized to any of the four molecular peanut allergens. In those who reported no history of peanut allergy, sensitization was seen in 0.7% (95% CI 0.

| Wheat
Molecular wheat allergens were evaluated in two studies. 39

Study design
Subjects participating

Study design
Subjects participating

Study design
Subjects participating

| Other allergens
In the study by Stemeseder et al., 42

| Period prevalence
In a birth cohort of 104 children from five German cities, Huang et al. 41 estimated the period prevalence of sensitization to Act d 2 during the age of 1-10 years to 7.7% (95% CI 3.9-14.5).

| Time trends
Huang et al. 41

| Summary of principal findings
This is the first systematic review to summarize the sensitization prevalence to molecular food allergens in the general population in Europe. Overall, sensitization was low to most of the 46 investigated molecular allergens. In non-allergic individuals, labile, cross-reactive molecular allergens were the most common, while individuals with food allergy were more commonly sensitized to storage proteins.
T A B L E 2 Assessment of risk of bias in the included studies F I G U R E 3 Point prevalence of sensitization to molecular peanut allergens investigated in two or more studies. Data from the red bars ("Peanut-allergic") are from Asarnoj et al., 37 and were estimated using ImmunoCAP™ (cut-off ≥ 0.35 kUA/l). Data from the "General population" are from Stemeseder et al., 41  Sensitization to 20 molecular allergens was non-existent or barely detected. In the general population, the highest sensitization was found for PR-10 proteins. There were too few studies to draw conclusions regarding which foods had the highest/lowest sensitization rates, and whether the "big eight" foods are the most common at the molecular allergen level. Some indications of regional differences were noted, such as lower sensitization to alpha-gal in Denmark than in Spain, but the data were insufficient to draw any conclusions, neither on country level nor on urban/rural level. It is also noteworthy, that while the included studies were published 2010-2018, the data were collected 2000-2014; thus, a significant proportion of the data describes sensitization prevalence from the early 21 st century.

| Comparison to previous and related literature
To the best of our knowledge, no systematic review has previously assessed the prevalence of sensitization to molecular allergens of multiple foods on a Europe-wide general population basis. Existing reviews have primarily focused on single foods in a non-systematic manner, 44,45 or on clinical aspects, such as sensitivity and specificity of molecular allergy diagnostic methods for certain foods. 46 The funders had no influence on the design of the study, interpretation of findings, or the decision to publish.